The present invention generally relates to detection of nucleic acid sequence mutations. The present invention more specifically relates to a new and useful method for signal amplification of mismatch cleavage.
The ability to detect alterations in nucleic acid sequences (for example, mutations and polymorphisms) is central to the diagnosis of genetic diseases and to the identification of clinically significant variants of disease-causing microorganisms. Similarly, identification and measurement of RNA is necessary for determining control of gene transcription.
One method for the molecular analysis of genetic variation involves the detection of restriction fragment length polymorphisms (RFLPs) using the Southern blotting technique (Southern, E. M., J. Mol. Biol., 98 503-517, 1975). Since this approach is relatively cumbersome, new methods have been developed, some of which are based on the polymerase chain reaction (PCR).
These include: RFLP analysis using PCR (Chehab et al., Nature, 329, 293-294, 1987; Rommens et al., Am. J. Hum. Genet., 46, 395-396, 1990), allele-specific amplification (ASA) (Newton C R et al., Nuc. Acids Res., 17, 2503-2516, 1989), oligonucleotide ligation assay (OLA) (Landergren U et al., Science 241, 1077-1080, 1988), primer extension (Sokolov B P, Nucl. Acids Res., 18, 3671, 1989), artificial introduction of restriction sites (AIRS) (Cohen L B et al., Nature 334, 119-121, 1988), allele-specific oligonucleotide hybridization (ASO) (Wallace R B et al., Nucl. Acids Res., 9, 879-895, 1981) and their variants.
The following are further examples of art discussing mismatch repair enzymes and systems utilizing such enzymes in addition to other related subject matter:
Lu et al., 80 Proc. Natl. Acad. Sci. USA 4639, 1983 disclose the use of a soluble E. coli system to support mismatch correction in vitro.
Pans et al., 163 J. Bact. 1007, 1985 disclose cloning of the mutS and mutL genes of Salmonella typhimurium. 
The specific components of the E. coli mispair correction system have been isolated and the biochemical functions determined. Preparation of MutS protein substantially free of other proteins has been reported (Su and Modrich, 1986, Proc. Nat. Acad. Sci. U.S.A., 84, 5057-5061. The isolated MutS protein was shown to recognize four of the eight possible mismatched base pairs (specifically, G-T, A-C, A-G and C-T mispairs).
U.S. Pat. No. 5,556,750 (xe2x80x9cMethods and kits for fractionating a population of DNA molecules based on the presence or absence of a base-pair mismatch utilizing mismatch repair systems, issued Sep. 17, 1996 to Duke University) describes methods of fractionating DNA molecules based on base-pair mismatch utilizing mismatch repair systems.
Su et al., 263 J. Biol. Chem. 6829, 1988 disclose that the mutS gene product binds to each of the eight base pair mismatches and does so with differential efficiency.
Jiricny et al., 16 Nucleic Acids Research 7843, 1988 disclose binding of the mutS gene product of E. coli to synthetic DNA duplexes containing mismatches to correlate recognition of mispairs and efficiency of correction in vivo. Nitrocellulose filter binding assays and band-shift assays were utilized.
Welsh et al., 262 J. Biol. Chem. 15624, 1987 purified the product of the MutH gene to near homogeneity and demonstrated the MutH gene product to be responsible for d(GATC) site recognition and to possess a latent endonuclease that incises the unmethylated strand of hemimethylated DNA 5xe2x80x2 to the G of d(GATC) sequences.
Au et al., 267 J. Biol. Chem. 12142, 1992 indicate that activation of the MutH endonuclease requires MutS, MutL and ATP.
Grilley et al. 264 J. Biol. Chem. 1000, 1989 purified the E. coli mutL gene product to near homogeneity and indicate that the mutL gene product interacts with MutS heteroduplex DNA complex.
Lahue et al., 245 Science 160, 1989 delineate the components of the E. coli methyl-directed mismatch repair system that function in vitro to correct seven of the eight possible base pair mismatches. Such a reconstituted system consists of MutH, MutL, and MutS proteins, DNA helicase II, single-strand DNA binding protein, DNA polymerase III holoenzyme, exonuclease I, DNA ligase, ATP, and the four deoxyribonucleoside triphosphates.
Su et al., 31 Genome 104, 1989 indicate that under conditions of restricted DNA synthesis, or limiting concentration of dNTPs, or by supplementing a reaction with a ddNTP, there is the formation of excision tracts consisting of single-stranded gaps in the region of the molecule containing a mismatch and a d(GATC) site.
Grilley et al. 268 J. Biol. Chem. 11830, 1993, indicate that excision tracts span the shorter distance between a mismatch and the d(GATC) site, indicating a bidirectional capacity of the methyl-directed system.
Holmes et al., 87 Proc. Natl. Acad. Sci. USA, 5837, 1990, disclose nuclear extracts derived from HeLa and Drosophila melanogaster K[c] cell lines to support strand mismatch correction in vitro.
Cooper et al., 268 J. Biol. Chem., 11823, 1993, describe a role for RecJ and Exonuclease VII as a 5xe2x80x2 to 3xe2x80x2 exonuclease in a mismatch repair reaction. In reconstituted systems such a 5xe2x80x2 to 3xe2x80x2 exonuclease function had been provided by certain preparations of DNA polymerase III holoenzyme.
Au et al., 86 Proc. Natl. Acad. Sci. USA 8877, 1989 describe purification of the mutY gene product of E. coli to near homogeneity, and state that the MutY protein is a DNA glycosylase that hydrolyzes the glycosyl bond linking a mispaired adenine (G-A) to deoxyribose. However, their enzyme did not cleave the A strand at xe2x80x9cAxe2x80x9d in a circular closed heteroduplex DNA with G/A mismatch as a substrate. The MutY protein, DNA polymerase I, and DNA ligase were shown to reconstitute G-A to G-C mismatch correction in vitro in the presence of an apurinic endonuclease.
Tsai-Wu et al., 89 Biochemistry 8779, 1992, cloned mutY gene, overexpress and purified the MutY enzyme to homogeneity for examining enzyme specificity. In addition to glycosylase activity, this MutY enzyme can cleave the xe2x80x9cAxe2x80x9d of G/A mismatch on the A strand.
Wiebauer and Jiricny, 339 Nature 234, 1989, discovered the correction of G/T mispairs to G/C pairs by thymine DNA glycosylase in human cells.
Nedderman and Jiricny, 268 J. Biol. Chem. 21218, 1993, purified the G/T mnispair specific thymine glycosylase from HeLa cells.
Slupsker et al., 178, J. Bacteriol. 3885, 1996, cloned and sequenced a human homolog (hMYH) of E. coli mutY gene whose function is the repair of oxidative DNA damage.
A role for the E. coli mismatch repair system in controlling recombination between related but non-allelic sequences has been indicated (Feinstein and Low, 113 Genetics, 13, 1986; Rayssiguier, 342 Nature 396, 1989; Shen, 218 Mol. Gen. Genetics 358, 1989; Petit, 129 Genetics 327, 1991). The frequency of crossovers between sequences which differ by a few percent or more at the base pair level are rare. In bacterial mutants deficient in methyl-directed mismatch repair, the frequency of such events increases dramatically. The largest increases are observed in MutS and MutL deficient strains. (Rayssiguier, supra; and Petit, supra.)
Nelson et al., 4 Nature Genetics 11, 1993, disclose a genomic mismatch (GMS) method for genetic linkage analysis. The method allows DNA fragments from regions of identity-by-descent between two relatives to be isolated based on their ability to form mismatch-free hybrid molecules.
During the last few years, a group of bacteria repair enzymes, e.g., endo VIII (Melamede, et al., Biochemistry, 33, 1255-1264, 1994), endo III (Dizdaroglu et al., Biochemistry, 32, 12105-12111, 1993), formamidopyrimidine-DNA glycosylase (Chetsanga et al., Biochemistry, 20, 5201-5207, 1981), T4 endonuclease V (Nakabeppu et al., J. Biol. Chem., 257, 2556-2562, 1982; McCullough et al., J. Biol. Chem., 272, 27210-27217, 1997), etc., have been isolated and characterized to repair the damaged and modified nucleic acid base. Like MutY and thymine glycosylase, these enzymes have a common repair mechanism of DNA N-glycosylase/AP lyases activity. The present invention is not limited to a specific repair enzyme. The use of these and other repair enzymes is contemplated by the invention and is consistent with the description below.
The present invention encompasses methods of amplification useful in the detection of specific genes, RNA transcripts and other DNA or RNA fragments. Specifically, the method of the invention is useful with detection methods which utilize labeled probes which bind with specificity to target molecules and which are complementary to the target with the exception of a mismatch site (e.g., the G/A or G/T basepairs) and which utilize a mismatch repair (MR) enzyme, which cleave only against the mismatch site of the double stranded complex.
For biological samples, the method of detection of the targets in genetic diseases or infectious microorganism needs a sensitivity of a million or less target DNA molecules in order to be practically useful.
In accordance with the invention, this high sensitivity is achieved by amplification of the products of the labeled probes cleaved from complementary DNA or RNA targets by mismatch repair (MR) enzymes, by recycling the target molecule. The assay temperature is set between the melting point (Tm) of the hybridized target/probe DNA duplex and that of the target/product complex. Upon cleavage of the probe by the MR enzyme, the invention utilizes amplifiers such as ammonium acetate (AA) or amiine derivatives (examples of which include, but are not limited to, diethylamine, piperidine and ammonium carbonate) to release the target DNA from the MR enzyme-target/product complex so that the target can again hybridize with intact probe molecules to form a target/probe duplex which is then also cleaved by MR enzyme and released from the MR enzyme. By the method of the invention, amplification of deoxyoligomer probe cleavage products results in greatly enhanced sensitivity.
The method of the invention, works similarly with RNA targets. Deoxyoligomer probes hybridize to target RNA to form a deoxyoligomer/RNA heterocomplex with a G/A mismatch site. The deoxyoligomer probe is cleaved at the xe2x80x9cAxe2x80x9d base of the mismatch site by the MR enzyme. The target RNA is freed from MR enzyme as described above for DNA targets and is so similarly able to again participate in target/probe hybridization. Amplification of the RNA signal by this method facilitates G/A basepair analysis of RNA transcripts in Northern analysis or retro viruses.
The various features of novelty which characterize the invention are pointed out with particularity in the claims annexed to and forming a part of this disclosure. For a better understanding of the invention, its advantages and objects, reference is made to the accompanying drawings and descriptive matter in which a preferred embodiment of the invention is illustrated.